Work on the development of instrumentation for the evaluation of various hemoglobin derivatives proposed as a blood substitute has proceeded at an accelerated rate with the coming on line of the pilot plant at LAIR which can furnish large amounts of the protein. The oxygen equilibrium curve apparatus is now being tested on human hemoglobin samples and calorimetric measurements of the binding of CO,CO2, and chloride to HbAo and the DBBF- HB product are underway. The pulsed accelerated flow apparatus is nearly ready for operation. Of considerable importance in evaluating the product's suitability for injection into humans is the purity of the material, a precise knowledge of the oxygen equilibrium curve and the protein's response to its various effectors, i.e. chloride, pH, temperature, phosphate, CO2, as well as oxygen concentration. Binding isotherms of oxygen and carbon monoxide in the presence and absence of these effectors are being determined both manometrically and optically as well as in the microcalorimeter. CO2 binding curves are being determined calorimetrically and by C13 NMR. The physical chemistry of hemoglobin has suffered in the past from the lack of a suitable standard state from which to start the free energy calculations. These studies on the isoionic protein, i.e. in the absence of all effectors, will allow such a standard state to be established.